Measuring Membrane Fluidity via Fluorescence

Membrane fluidity can be measured as a fluorescence polarization or anisotropy value, which corresponds to how a fluorescent probe inside the membrane reacts to polarized light (Mykytczuk et al. 2007 (link)). Harvested cells were treated according to the protocol described by Beney et al. (2004 (link)). Briefly, the samples were washed twice in PBS, pH = 7.0, resuspended (1 × 10⁸ cells/ml), and incubated at 37 °C for 30 min with 1,6-diphenyl-1,3,5-hexatriene (DPH; supplied by Life Technologies, Carlsbad, CA, USA) at a concentration of 0.2 μM (0.2 mM stock solution in tetrahydrofuran). Fluorescence polarization values were determined by using a Synergy 2 Multi-Mode microplate reader from BioTek using sterile black-bottom Nunclon delta surface 96-well plates. The filters were 360/40 nm fluorescence excitation and 460/40 nm fluorescence emission filters from BioTek. The excitation polarized filter was set in the vertical position. The emission polarized filter was set either in the vertical (I_VV) or horizontal (I_VH) position. The polarization value is calculated by the following formula: $P=\frac{I_{\mathrm{VV}}-I_{\mathrm{VH}}G}{I_{\mathrm{VV}}+I_{\mathrm{VH}}G},$ where G is the grating factor, assumed to be 1. The cells were treated with octanoic acid at pH 7.0 just before measurements.